Ionotropic glutamate receptors (GluRs) are molecular pores which facilitate the passage of ions across cell membranes and mediate excitatory signal transmission in the mammalian nervous system. Because of their essential role in normal brain function and development and increasing evidence that dysfunction of GluR activity mediates multiple CNS diseases as well as damage during stroke a substantial effort has been directed towards analysis of GluR properties. Surprisingly, GluRs are also present in bacteria, where their function is unknown. The AMPA, kainate and NMDA subtypes of eukaryotic ionotropic glutamate receptors are encoded by 7 gene families. The recent crystallization of the ligand binding core of an AMPA receptor subunit has revealed for the first time the molecular mechanisms underlying the binding of agonists and antagonists and current work is directed towards structural studies on other receptor subtypes. Structural studies show that, despite their specialized role in brain function, glutamate receptors appear to have evolved directly from bacterial ion channels. Direct evidence was obtained for this from the identification and analysis of the first GluR found in a prokaryote: GluR0 from the photosynthetic bacterium syncheocystis PCC 6803. GluR0 binds glutamate, forms potassium-selective channels, and is related in amino acid sequence to both eukaryotic GluRs and potassium channels. On the basis of the amino acid sequence and functional relationships between GluR0 and eukaryotic GluRs, it seems likely that a prokaryotic GluR was the precursor to eukaryotic GluRs. We have found that GluR0 can be gated not only by glutamate but also by external protons or by lowering the Ca2+ concentration of the external solution. The single-channel responses gated by either protons or by lowering Ca2+ exhibited a voltage-dependent block by external Na+, which was similar to that of desensitizing responses to glutamate. The single-channel activity gated by all three conditions typically displayed two types of gating behavior: long open times interspersed with fast flickering bursts. Proton gated currents, EC50 30 mM, exhibited a pH-dependent block of single channel conductance at -60 mV and were cross-desensitized by 1mM glutamate. Similarly, GluR0 responses activated by lowering extracellular calcium but not magnesium to 10 nM at pH 7.2, IC50 10 uM, were desensitized by 1mM glutamate. The independence of these gating mechanisms was demonstrated by a binding site mutant, GluR0/R117K, which maintained normal pH and Ca2+ sensitivity while virtually abolishing activation by glutamate. Although the gating of many ion channels can be modulated by pH or by Ca2+ GluR activation by protons in the absence of ligand and spontaneous activity inhibited by Ca2+ has not been described before. The ligand binding core of GluR0 has been purified and over expressed. High resolution structures of the ligand binding core of GluR0, the glutamate receptor ion channel from Synechocystis PCC 6803, have been solved by X-ray diffraction. The GluR0 structures reveal homology with bacterial periplasmic binding proteins and the rat GluR2 AMPA subtype neurotransmitter receptor. The ligand binding site is formed by a cleft between two globular alpha/beta domains. L-glutamate binds in an extended conformation, similar to that observed for glutamine binding protein (GlnBP). However, the L-glutamate gamma-carboxyl group interacts exclusively with Asn51 in domain 1, different from the interactions of ligand with domain 2 residues observed for GluR2 and GlnBP. To address how neutral amino acids activate GluR0 gating we solved the structure of the binding site complex with L-serine. This revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group makes solvent mediated hydrogen bonds with Asn51. The structure of a ligand-free, closed-cleft conformation revealed an extensive hydrogen bond network mediated by solvent molecules. Equilibrium centrifugation analysis revealed dimerization of the GluR0 ligand binding core with a dissociation constant of 0.8 ?M. In the crystal, a symmetrical dimer involving residues in domain 1 occurs along a crystallographic 2-fold axis and suggests that tetrameric glutamate receptor ion channels are assembled from dimers of dimers. We propose that ligand induced conformational changes cause the ion channel to open as a result of an increase in domain 2 separation relative to the dimer interface. The binding of agonists to glutamate receptors triggers ion channel gating which within milliseconds is followed by desensitization. The molecular mechanisms of desensitization are unknown. In collaboration with the Gouaux lab at HHMI Columbia University we have performed functional studies to address the molecular mechanisms of gating. Mutations have been identified which alter the extent and kinetics of desensitization. These map to a surface of the ligand binding core which crystallographic studies reveal to mediate interactions between GluR subunit dimers. Analytical ultracentrifugation was used to measure the Kd for dimerization of GluR2 ligand binding cores in solution. A piezo concentration jump apparatus was used to measure desensitization in outside out patches. Mutants which increase dimer stability reduce desensitization, while mutants which decrease dimer stability increase desensitization. Crystallographic analysis of the non desensitizing GluR2 mutant L748Y reveals that the Tyrosine side chain in one subunit interacts with a pocket in the opposite subunit of the dimer pair. Mutation to alanine of the side chains which form this pocket restores desensitization and increases the Kd for dimer formation. We propose that movement of GluR subunits about the dimer interface allow the ion channel to close even though the agonist binding domains remain in their closed-cleft -bound conformation which initially promotes ion channel gating. Our results reveal for the first time a molecular mechanism for GluR receptor desensitization.